In the field of therapeutics the use of proteins and antibodies and antibody-derived molecules in particular has been constantly gaining presence and importance, and, consequently, the need for controlled manufacturing processes has developed in parallel. The commercialization of therapeutic proteins requires they are produced in large amounts. For this purpose the protein is frequently expressed in a host cell and subsequently be recovered and purified, prior to its preparation into an administrable form.
Depending on the protein to be expressed the choice of host cell may be a mammalian host cell, frequently a CHO (Chinese hamster ovary) cell, or a bacterial host cell. In the first case, the protein is typically secreted into the culture supernatant which is recovered, and the solution is then processed for protein purification.
When the host cell is a Gram negative prokaryotic cell, an often preferred expression system involves the newly synthesized protein accumulating within and being isolated from the periplasmic space. In this case, once the desired level of protein expression has been achieved, it is the cells that are harvested and processed. The protein is then recovered by means of subjecting the harvested cells to a protein extraction process which involves releasing the protein from the periplasm into solution and subsequent removal of cell debris and other impurities. These steps of cell harvest to protein release are typically included in what is termed primary recovery. The resulting protein-containing solution is then processed for protein purification. Preferred Gram negative prokaryotic cells used for periplasmic expression are generally Escherichia coli strains or Pseudomonas fluorescens cells.
Protein purification from complex mixtures is adapted to the target protein. In the case of antibodies and antibody-derived products, purification typically involves a first product capture step via chromatography which offers a first purification and significant concentration of the product. This first step is usually followed by one or more further chromatographic steps used to reduce contaminants such as host cell impurities, medium, purification process-related impurities and product-related impurities.
In recent years it has become increasingly common for different proteins, including antibodies and antibody derived fragments, to be coupled to another molecule with a particular function, this is applied both to diagnostic and therapeutic uses, to name but a few examples it may be to target the antibody to a particular set of cells, alternatively it may be a drug which is targeted by the antibody to its specific site of action, or the molecule may be destined to increase the antibody's half-life in an animal. The latter is particularly the case regarding antigen binding antibody fragments which tend to be rapidly cleared from the circulation of animals.
Different molecules can be coupled to a protein via a reactive group in the protein which either occurs naturally in the protein or is artificially introduced by protein engineering. Frequently, favoured reactive groups for protein binding to a second molecule are thiol groups present in unpaired cysteine residues. In this sense, antibody hinges are common regions for site specific reaction since they contain cysteine residues and are remote from other regions of the antibody likely to be involved in antigen binding. For example, reaction with polyethylene glycol (PEG) or PEGylation of thiol groups is a well-known approach to site-specific PEGylation, where a variety of thiol-specific reagents are available.
However, in native proteins, cysteine residues are usually involved in disulfide bridges or responsible for interaction with metals or other proteins. Therefore, in order to achieve site-specific binding, these reactive groups need to be in the correct conformation, i.e. their thiol groups being free, so as to enable their reaction. For example, PEGylation of G-CSF at a single cysteine residue was described in Veronese et al. Bioconjugate Chemistry 2007 November-December; 18(6):1824-30 whereby the PEGylation was performed under transient denaturing conditions. There have also been attempts in the prior art to optimise processes to obtain the protein in an optimal conformation to enable its subsequent reaction to the desired molecule. For example, WO 2007/003898 describes a specific diareduction step incorporated after purification of a Fab′ to prepare it for subsequent reaction with PEG. In many cases, such as with a Fab′, it is desirable to selectively affect one or more target cysteines for conjugation without reducing other cysteines present in the protein. For example in this case, Fab's have a native interchain disulphide bond between the heavy and light chain constant regions and so in order to selectively reduce a target cysteine elsewhere in the antibody, e.g. the hinge, reduction needs to be carried out with care so the disulphide chain remains intact and reaction with the interchain cysteines is avoided. Typically, this is achieved using what are considered to be mild reducing conditions. However, given that reduction is a common chemical reaction, even under said mild reducing conditions, different compounds present in a complex mixture are capable of reacting in a reducing environment resulting in altered properties, such as but not limited to binding behaviours, that can affect subsequent protein purification. For this reason reduction steps described in the prior art are performed once the protein has been purified. Subsequently, the desired protein conjugate must be purified from unreacted protein or other undesired conjugates. The efficiency of this coupling reaction process relates directly to production efficiency and associated manufacturing costs.
Given the above, there is a further need in the art to provide improved methods for protein manufacture, particularly where the manufactured protein is to be coupled to a second molecule.